Canola variety producing a seed with reduced glucosinolates and linolenic acid yielding an oil with low sulfur, improved sensory characteristics and increased oxidative stability

ABSTRACT

A canola line has been stabilized to produce seeds having an α-linolenic acid content of less than that of generic canola oil, more preferably less than or equal to about 7% α-linolenic acid relative to total fatty acid content of said seed and a total glucosinolate content of less than 18 μmol/g of defatted meal, more preferably less than or equal to about 15 μmol/g of defatted meal. This canola line has reduced sulfur content of less than or equal to 3.0 ppm, improved sensory characteristics and increased oxidative stability.

TECHNICAL FIELD

[0001] This invention relates to improved canola seeds, plants and oilhaving advantageous properties, that is, a low glucosinolates contentand a very low α-linolenic acid (C_(18:3)) content, which produce an oilwith low sulfur content, improved sensory characteristics and oxidativestability.

BACKGROUND

[0002] A need exists for an improved vegetable oil with a significantlyextended shelf life and greater heat stability relative to genericcanola oil and a positive nutritional contribution to animal, includinghuman, diets.

[0003] Canola oil has the lowest level of saturated fatty acids of allvegetable oils. “Canola” refers to rapeseed (Brassica) which has anerucic acid (C_(22:1)) content of at most 2 percent by weight based onthe total fatty acid content of a seed, preferably at most 0.5 percentby weight and most preferably essentially 0 percent by weight and whichproduces, after crushing, an air-dried meal containing less than 30micromoles (μmol) per gram of defatted (oil-free) meal. These types ofrapeseed are distinguished by their edibility in comparison to moretraditional varieties of the species.

[0004] As consumers become more aware of the health impact of lipidnutrition, consumption of canola oil in the U.S. has increased. However,generic canola oil cannot be used in deep frying operations, animportant segment of the food processing industry.

[0005] Canola oil extracted from natural and previously commerciallyuseful varieties of rapeseed contains a relatively high (8%-10%)α-linolenic acid content (C_(18:3)) (ALA). This trienoic fatty acid isunstable and easily oxidized during cooking, which in turn createsoff-flavors of the oil (Gailliard, 1980, Vol. 4, pp. 85-116 In: Stumpf,P. K., ed., The Biochemistry of Plants, Academic Press, New York). Italso develops off odors and rancid flavors during storage (Hawrysh,1990, Stability of canola oil, Chapter 7, pp. 99-122 In: F. Shahidi, ed.Canola and Rapeseed: Production, Chemistry, Nutrition, and ProcessingTechnology, Van Nostrand Reinhold, New York). One such unsatisfactoryspecies heretofore has been Brassica napus, i.e., spring canola, a typeof rapeseed.

[0006] It is known that reducing the α-linolenic content level byhydrogenation increases the oxidative stability of the oil.Hydrogenation is routinely used to reduce the polyunsaturates content ofvegetable oils, thereby increasing its oxidative stability. The foodindustry has used hydrogenation to raise the melting point of vegetableoils, producing oil-based products with textures similar to butter, lardand tallow. Trans isomers of unsaturated fatty acids are commonlyproduced during hydrogenation. However, the nutritional properties oftrans fatty acids mimic saturated fatty acids, thereby reducing theoverall desirability of hydrogenated oils (Mensink et al., New EnglandJ. Medicine N323:439-445, 1990; Scarth, et al., Can. J. Pl. Sci.,68:509-511, 1988). Canola oil produced from seeds having a reducedα-linolenic acid content would be expected to have improvedfunctionality for cooking purposes with improved nutritional value, andtherefore have improved value as an industrial frying oil.

[0007] However, in general, very little variation exists for α-linolenicacid content in previously known canola quality B. napus germplasm(Mahler et al., 1988, Fatty acid composition of Idao Misc. Ser. No.125). Lines with levels of α-linolenic acid lower than that of genericcanola oil are known, but have sensory, genetic stability, agronomic orother nutritional deficiencies. For example, Rakow et al. (J. Am. OilChem. Soc., 50:400-403, 1973), and Rakow (Z. Pflanzenzuchtg, 69:62-82,1973), disclose two α-linolenic acid mutants, M57 and M364, produced bytreating rapeseed with X-ray or ethylmethane sulfonate. M57 had reducedα-linolenic acid while M364 had increased α-linolenic acid. However, theinstability of the fatty acid traits between generations wasunacceptable for commercial purposes.

[0008] Brunklaus-Jung et al. (Pl. Breed., 98:9-16, 1987), backcrossedM57 and other rapeseed mutants obtained by mutagenic treatment tocommercial varieties. BC₀ and BC₁ of M57 contained 29.4-33.3% oflinoleic acid (C_(18:2)) and 4.9-10.8% of α-linolenic acid (C_(18:3)).The oleic acid (C_(18:1)) content was not reported, but by extrapolationcould not have exceeded 60%.

[0009] Four other lower α-linolenic acid canola lines have beendescribed. Stellar, reported by Scarth et al. (Can. J. Plant Sci.,68:509-511, 1988), is a Canadian cultivar with lower α-linolenic acid(also 3%) derived from M57. Its α-linolenic acid trait was generated byseed mutagenesis. S85-1426, a Stellar derivative with improved agronomiccharacteristics, also has lower (1.4%) α-linolenic acid (Report of 1990Canola/Rapeseed Strain Test A, Western Canada Canola RapeseedRecommending Committee). IXLIN, another lower α-linolenic acid (1.8%)line described by Roy et al. (Plant Breed., 98:89-96, 1987), originatedfrom an interspecific selection. EP-A 323 753 (Allelix) discloses rapeplants, seeds, and oil with reduced α-linolenic acid content linked tolimitations in the content of oleic acid, erucic acid, andglucosinolate.

[0010] Another nutritional aspect of rapeseed, from which canola wasderived, is its high (30-55 μmol/g) level of glucosinolates, asulfur-based compound. When the foliage or seed is crushed,isothiocyanate esters are produced by the action of myrosinase onglucosinolates. These products inhibit synthesis of thyroxine by thethyroid and have other anti-metabolic effects (Paul et al., Theor. Appl.Genet. 72:706-709, 1986). Brassica varieties with reduced glucosinolatescontent (<30 μmol/g defatted meal) were developed to increase thenutritional value of canola meal (Stefansson et al., Can. J. Plant Sci.55:343-344, 1975). Meal from an ultra-low glucosinolates line, BC86-18,has 2 μmol/g total glucosinolates and significantly improved nutritionalquality compared to generic canola meal (Classen, Oral presentation,GCIRC Eighth International Rapeseed Congress, Saskatoon, Saskatchewan,Jul. 9-11, 1991). Neither its fatty acid composition nor its seedglucosinolates profile is known.

[0011] There remains a need for an improved canola seed and oil withvery low α-linolenic levels in the oil and low glucosinolates in theseed to significantly reduce the need for hydrogenation. The α-linoleniccontent of such a desirable oil would impart increased oxidativestability, thereby reducing the requirement for hydrogenation and theproduction of trans fatty acids. The reduction of seed glucosinolateswould significantly reduce residual sulfur content in the oil. Sulfurpoisons the nickel catalyst commonly used for hydrogenation (Koseoglu etal., Chapter 8, pp. 123-148, In: F. Shahidi, ed. Canola and Rapeseed:Production, Chemistry, Nutrition, and Processing Technology, VanNostrand Reinhold, New York, 1990). Additionally, oil from a canolavariety with low seed glucosinolates would be less expensive tohydrogenate.

SUMMARY OF THE INVENTION

[0012] This invention comprises a Brassica napus canola yielding seedhaving a total glucosinolates content of about 18 μmol/g or less ofdefatted, air-dried meal; the seed yielding extractable oil having 1) anα-linolenic acid content of about 7% or less relative to total fattyacid content of the seed, and 2) a very low sulfur content of less thanor equal to 3.00 ppm. The invention also includes a Brassica napusyielding canola oil having, when hydrogenated, a significantly reducedoverall room-odor intensity relative to the overall room-odor intensityof generic canola oil. The new variety more particularly yieldsnon-hydrogenated oil significantly reduced in fishy odor relative to thefishy odor of generic canola oil, such odor being characteristic ofBrassica seed oil. The seed of such canola variety has an α-linolenicacid content of less than or equal to 7%, more preferably less than orequal to about 4.1% (α-linolenic acid (C_(18:3)) relative to total fattyacid content of said seed and a total glucosinolates content of lessthan 18 μmol/g, more preferably less than or equal to about 15 μmol/gand most preferably less than or equal to 13 μmol/g and belongs to aline in which these traits have been stable for both the generation towhich the seed belongs and that of its parent.

[0013] This invention further includes processes of making crosses usingIMC 01 as at least one parent of the progeny of the above-describedseeds and oil derived from said seeds.

[0014] This invention further comprises a seed designated IMC 01deposited with the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md., USA 20852 and bearing accession number ATCC40579, the progeny of such seed and oil of such a seed possessing thequality traits of interest.

DETAILED DESCRIPTION OF THE INVENTION

[0015] A spring canola (Brassica napus L.) variety was developed withimproved sensory characteristics and oxidative stability in the seedoil. This variety, designated IMC 01, has very low levels of α-linolenicacid (CH₃CH₂CH═CHCH₂CH═CHCH₂CH═CH (CH₂)₇COOH) in the seed oil and verylow levels of glucosinolates in the seed. The oil produced from the seedof this variety has very low levels of sulfur and was shown to havesignificantly improved sensory characteristics over generic canola oils.The IMC 01 is a line in which these traits have been stabilized for boththe generation to which the seed belongs and that of its parentgeneration. Particularly desirable lines of this invention from anagronomic point of view can be derived by conventionally crossing linesproducing seeds meeting the definitions of this invention withagronomically well-proven lines such as Westar.

[0016] In the context of this disclosure, a number of terms are used. Asused herein, a “line” is a group of plants that display little or nogenetic variation between individuals for at least one trait. Such linesmay be created by several generations of self-pollination and selection,or vegetative propagation from a single parent using tissue or cellculture techniques. As used herein, the terms “cultivar” and “variety”are synonymous and refer to a line which is used for commercialproduction. “Stability” or “stable” means that with respect to the givencomponent, the component is maintained from generation to generationand, preferably, at least three generations at substantially the samelevel, e.g., preferably ±15%, more preferably ±10%, most preferably ±5%.The stability may be affected by temperature, location, stress and thetime of planting. Comparison of subsequent generations under fieldconditions should produce the component in a similar manner. “CommercialUtility” is defined as having good plant vigor and high fertility, suchthat the crop can be produced by farmers using conventional farmingequipment, and the oil with the described components can be extractedfrom the seed using conventional crushing and extraction equipment. Tobe commercially useful, the yield, as measured by both seed weight, oilcontent, and total oil produced per acre, is within 15% of the averageyield of an otherwise comparable commercial canola variety without thepremium value traists grown in the same region. “Agronomically elite”means that a line has desirable agronomic characteristics such as yield,maturity, disease resistance, standability. The amount of fatty acids,such as oleic and linolenic acids, that are characteristic of the oil isexpressed as a percentage of the total fatty acid content of the oil.“Saturated fatty acid” refers to the combined content of palmitic acidand stearic acid. “Polyunsaturated fatty acid” refers to the combinedcontent of linoleic-and α-linolenic acids. The term “shortening” refersto an oil that is a solid at room temperature. The term “room odor”refers to the characteristic odor of heated oil as determined using theroom-odor evaluation method described in Mounts (J. Am. Oil Chem. Soc.,56:659-663, 1979). “Generic canola oil” refers to a composite oilextracted from commercial varieties of rapeseed currently known as ofthe priority date of this application, which varieties generallyexhibited at a minimum 8-10% α-linolenic acid content, a maximum of 2%erucic acid and a maximum of 30 μmol/g total glucosinolate level. Theseed from each growing region is graded and blended at the grainelevators to produce an acceptably uniform product. The blended seed isthen crushed and refined, the resulting oil being sold for use. Table Ashows the distribution of canola varieties seeded as percentage of allcanola seeded in Western Canada in 1990. TABLE A Distribution of CanolaVarieties Grown in Western Canada in 1990 Percent of Canola VarietySeeded Area B. campestris Candle 0.4 Colt 4.4 Horizon 8.5 Parkland 2.5Tobin 27.1 B. napus Alto 1.1 Delta 0.9 Global 0.9 Legend 18.2 Pivot 0.1Regent 0.5 Stellar 0.2 Tribute 0.4 Triton 0.7 Triumph 0.2 Westar 29.5Others 4.4

[0017] Source: Quality of Western Canadian Canola—1990 Crop Year. Bull.187, DeClereg et al., Grain Research Laboratory, Canadian GrainCommission, 1404-303 Main Street, Winnipeg, Manitoba, R3C 3G8.

[0018] IMC 01 is a very low α-linolenic acid (<4.1% C_(18:3)) lineselected during an extensive germplasm screening effort. Its parentageis unknown. IMC 01 was self-pollinated and selected for low α-linolenicacid (<4.1%) over four consecutive generations. At each generation,seeds from individually pollinated plants were analyzed for fatty acidcomposition. Data showed no genetic segregation for α-linolenic acidcontent over five generations of self-pollinations (Table I). Breederseed was derived from a bulk seed increase of selected plants from thefourth self-crossed generation. TABLE I Fatty Acid Composition of IMC 01Over Five Generations PERCENT COMPOSITION DATE OF ANALYSIS C_(16:0)C_(18:0) C_(18:1) C_(18:2) C_(18:3) December 1987 4.1 1.9 64.1 25.7 1.9August 1988 4.6 2.3 72.6 14.4 2.0 January 1989 4.9 1.5 60.4 25.8 2.5April 1989 4.8 1.8 64.3 21.4 4.0 November 1989 4.3 2.1 64.1 24.8 2.0

[0019] IMC 01 was planted-in replicated field trials in North Dakota,South Dakota, Minnesota, Washington, Idaho and Montana in 1989 and 1990,under both irrigated and nonirrigated conditions. These tests showedthat the α-linolenic acid content of IMC 01 was sensitive to temperature(Table II). This was further supported by growing IMC 01 undercontrolled temperature conditions in growth chambers. Whether or not theobserved temperature sensitivity of IMC 01 is common to other lowα-linolenic acid canola lines is unknown.

[0020] A temperature effect on fatty acid compositions has been widelyreported in plants, especially oilseed crops (Rennie et al., J. Am. OilChem. Soc., 66:1622-1624, 1989). These reports describe generaltemperature effects on fatty acid composition.

[0021] Changes in fatty acid content in seed oil under cool temperatureshave been documented in plants such as soybean, peanut and sunflower(Neidleman, In: Proceedings of the World Conference on Biotechnology forthe Fats and Oils Industry, Applewhite, T. H., ed., pp. 180-183. Am.Oil. Chem. Soc. 1987). TABLE II α-Linolenic acid Content of IMC 01 inProduction in 1990 α-LINOLENIC PRODUCTION {overscore (X)} TEMPERATUREDURING ACID REGION SEED MATURATION CONTENT Eastern Washington 74° F.1.9% Eastern Washington 74° F. 2.0% Northern Idaho 70° F. 3.0% NorthernIdaho 67° F. 2.9% Eastern Idaho 62° F. 3.5% Southern Montana 66° F. 4.1%Central Montana 67° F. 4.0%

[0022] In addition to very low α-linolenic acid, IMC 01 is alsocharacterized by very low levels of glucosinolates. Glucosinolates aresulfur-based compounds common to all Brassica seeds. Glucosinolatesexist in aliphatic or indolyl forms. Aliphatic glucosinolates can beanalyzed via gas chromatography (GC) (Daun, Glucosinolate analysis ofrapeseed (canola), Method of the Canadian Grain Commission, GrainResearch Laboratory, Canadian Grain Commission, Winnipeg, 1981). Indolylglucosinolates have only recently been analyzed via high performanceliquid chromatograph (HPLC). Prior to the adoption of the HPLC method,total glucosinolates were calculated by multiplying the aliphaticglucosinolates by a correction factor. Canola quality in the seed isdefined as having <30 μmol/g of glucosinolates in the defatted meal.

[0023] IMC 01 and Westar were tested in five locations in southeasternIdaho in 1990. Three of the locations were irrigated (I) and two weredryland (D) conditions. Table IIIa shows the difference in totalaliphatic glucosinolate between IMC 01 and Westar grown at theselocations. The aliphatic glucosinolate values are reported as μmol/gm ofdefatted meal.

[0024] The aliphatic glucosinolate content of IMC 01 by location wasconsistently lower and more stable than that of Westar at all locationstested. The average glucosinolate contents of IMC 01 was 4.9 μMol/gmwhile Westar was 13.3 μmol/gm. A Least Significant Difference (LSD) testwas used to determine if the two were significantly different at alllcoations. IMC 01 was found to be significantly different from Westar ata level of P<0.05.

[0025] HPLC analysis of IMC 01 vs Westar, the most widely grown springcanola variety in North America, shows that IMC 01 has much lower levelsof aliphatic glucosinolates (Table III). No significant differencesexist for indolyl glucosinolates. Glucosinolates content is also subjectto environment influence, e.g., sulfur fertility and drought stress.However, IMC 01 consistently had the lowest and the most stablealiphatic glucosinolates levels at all locations tested (Table IV). Thelocations tested differ in altitude, temperature, fertility, irrigation,and other cultural practices. Among the low α-linolenic canola lines forwhich glucosinates analysis have been performed, IMC 01 has the lowestlevel of total seed glucosinolates (Table V). TABLE III GlucosinolatesProfiles of IMC 01 and Westar Varieties GLUCOSINOLATES (μmol/g) IMC 01WESTAR 3-butenyl 1.2 4.2 4-pentenyl 0.1 0.2 2-OH-3-butenyl 3.1 7.02-OH-4-pentenyl 0.9 0.4 Total Aliphatics 5.3 11.8  4-OH-3-indolylmethyl6.2 6.1 3-indolylmethyl 0.8 1.0 4-methoxyindolyl 0.1 tr 1-methoxyindolyl0.1 0.2 Total Indolyls 7.2 7.3 Total Glucosinolates 12.5 19.1 

[0026] TABLE IV Aliphatic Glucosinolates of IMC 01 and Westar overDifferent Environments in Southeastern Idaho ALIPHATIC GLUCOSINOLATESCONTENT (μmol/g) LOCATION* IMC 01 WESTAR Newdale - I 4.7 13.0 SodaSprings - D 6.3 9.9 Tetonia - D 5.0 13.5 Tetonia - I 3.7 13.3 Shelley -I 5.0 17.0 Average 4.9 13.3 Standard Deviation 0.93 2.51

[0027] TABLE V Glucosinolates Content of Low α-Linolenic acid CanolaVarieties and Westar GLUCOSINOLATES (μmol/g) ALIPHATIC INDOLYL TOTAL IMC01 5.3 7.2 12.5 Stellar 5.2 19.5 24.7 S85-1426 7.9 13.4 21.3 Westar 11.87.3 19.1

[0028] IMC 01 was produced, using normal production practices for springcanola, in Idaho and North Dakota in 1988, in Idaho, Washington Stateand Montana in 1989, in Idaho, Washington State, Montana, Oregon, andWyoming in 1990. When grown in suitable environments, where the averagedaily temperature (high temperature plus low temperature divided by 2)exceeds 20° C., the oil contains <4.1% α-linolenic acid. As an example,a normal fatty acid profile was produced at Casselton, N. Dak. The cropproduced in Ashton, Id., was subject to extremely cool conditions andhad higher levels of α-linolenic acid. The crops obtained from the fieldtests were crushed and processed to produce refined, bleached anddeodorized (RBD) canola oil at the Protein, Oil, Starch (POS) PilotPlant in Saskatoon, Saskatchewan. A method of bleaching canola oil isprovided in the AOCS' Recommended Practice Cc 8a-52 (AOCS Methods andStandard Practices, 4th Edition (1989)). A method for the refining ofcrude oils is provided in the AOCS Practice Cc 9a-52 (AOCS Methods andStandard Practices, 4th Edition (1989)). The oils were tested at theVegetable Oil Research Laboratory, U.S.D.A./Northern Regional ResearchCenter, for organoleptic and sensory characteristics.

[0029] Testing to assure that desirable sensory characteristics areobtained in the Brassica napus variety was essential. The evaluation ofodors has been conducted in a variety of ways on low α-linolenic acidcanola oils. The testing methods are based on the fact that vegetableoils emit characteristic odors upon heating. For example, Prevot et al.(J. Amer. Oil Chemists Soc. 67:161-164, 1990) evaluated the odors of aFrench rapeseed, “Westar”, and “low linolenic” canola oils in a testwhich attempted to reproduce domestic frying conditions. In theseevaluations the test oils were used to fry potatoes and the odors wereevaluated by a test panel. The odor tests showed that the “lowlinolenic” (approximately 3%) line had a significantly higher (moreacceptable) odor score than the French rapeseed and “Westar” lines,which were very similar to each other. Eskin et al. (J. Amer. OilChemists Soc. 66:1081-1084, 1989) evaluated the odor from canola oilwith a low linolenic acid content, a laboratory deodorized sample, and acommercially deodorized sample by sniffing in the presence of the oilitself. These studies demonstrated that a reduction in the linolenicacid content of canola oil from 8-9% to 1.6% reduced the development ofheated odor at frying temperatures. However, the odor of the lowlinolenic acid oil was still unacceptable when heated in air to amajority of the panelists, suggesting that low linolenic acid alone isnot sufficient to guarantee acceptable odor.

[0030] Mounts (J. Am. Oil Chem. Soc., 56:659-663, 1979) describe adistinct room-odor evaluation method that is used to reproducibly assessthe odor characteristics of a cooking oil upon heating. This is theevaluation method of choice owing to its reproducibility and itsapproximation of odors emitted upon heating the oil. In this method, theoil is heated in a separate chamber and the odor pumped into the roomcontaining the trained evaluators. As noted elsewhere, where the term“room-odor” is used herein, it refers to this method of Mounts. Thismethod is distinct from earlier described tests where the oil andevaluator are within the same room. Such same room testing is referredto as “uncontrolled bench top odor tests” and is considered lessaccurate and less reliable than the Mounts' room odor evaluation method.

[0031] The room-odor characteristics of cooking oils can be reproduciblycharacterized by trained test panels in room-odor tests (Mounts, J. Am.Oil Chem. Soc. 56:659-663, 1979). A standardized technique for thesensory evaluation of edible vegetable oils is presented in AOCS'Recommended Practice Cg 2-83 for the Flavor Evaluation of Vegetable Oils(Methods and Standard Practices of the AOCS, 4th Edition (1989)). Thetechnique encompasses standard sample preparation and presentation, aswell as reference standards and method for scoring oils. When heated,generic canola oil has reduced stability and produces offensive roomodors. Refined-Bleached-Deodorized (RBD) canola oil is characterized bya fishy flavor in such tests. This characteristic is commonly ascribedto its high polyunsaturated fatty acid content, particularly α-linolenicacid, relative to other vegetable oils. The individual fragrance notes(odor attributes) of the oils are evaluated by Least SignificantDifference Analysis. Notes which differ by greater than 1.0 can bereproducibly measured by a sensory panel. In these tests, IMC 01 oilexpressed significantly reduced levels of the offensive odors (TableVI). TABLE VI Room Odor Intensity of IMC 01 and Generic Canola OilGENERIC ODOR ATTRIBUTES IMC 01 CANOLA OIL Overall 4.6^(a) 7.4^(b) FriedFoods 1.8 3.5 Doughy 1.0 0 Fishy 0^(a) 5.5^(b) Burnt 0^(a) 0.9^(b) Acrid0^(a) 2.3^(b) Woody/Cardboard 1.9 0 Hydrogenated 0 0 Pastry/Sugary 0 0Waxy 0 0 Chemical 0 0

[0032] Due to its relatively low stability, canola oil is oftenhydrogenated for frying. However, hydrogenation produces acharacteristic (hydrogenated) room odor which is unacceptable to foodmanufacturers. Surprisingly, hydrogenated IMC 01 oil also has reducedlevels of the characteristic hydrogenated room odor (Table VII). TableVII shows that the overall room-odor intensity of hydrogenated IMC 01 issignificantly less than that of hydrogenated generic oil as indicated bya difference is scores of greater than 1.0 in standardized flavorevaluation trials. TABLE VII Room Odor Intensity and Individual OdorDescriptions for Hydrogenated Canola Oils HYDROGENATED HYDROGENATED IMC01 HYDROGENATED, IMC 01 SHORTENING GENERIC SHORTENING (2% (6.8% CANOLAα-LINOLENIC α-LINOLENIC ODOR ATTRIBUTE SHORTENING ACID) ACID) OverallIntensity 6.6^(b) 3.8^(a) 3.9^(a) Fried Food 2.7 1.1 1.5 Doughy 1.2 0.60.8 Fishy 0.6 0 0 Burnt 0.5 0 0 Acrid 0.8 0 0 Hydrogenated 3.2 1.8 2.3Waxy 0.5 0.6 0. Other 4.5 2.4 2.8 rubbery fruity fruity flowery smokyflowery weedy sweet soapy pastry

[0033] IMC 01 produces an oil which has improved sensorycharacteristics. Such improvements have been predicted for lowα-linolenic acid canola oils (Ulrich et al., J. Am. Oil Chem. Soc.,8:1313-1317, 1988). However, the improved sensory characteristics of IMC01 appears not to be related solely to its low α-linolenic acid content.Surprisingly, IMC 01 canola oils with both high and low levels ofα-linolenic acid showed similar degrees of improvement. Sensory testshave shown that IMC 01 oil maintains its improved quality at both 2% and6.8% α-linolenic acid.

[0034] The very low glucosinolates characteristic of IMC 01 seed isbelieved to contribute to the improved sensory characteristic of IMC 01oil. Glucosinolates in the seed are converted to sulfur compounds. Mostof the sulfur breakdown products remain in the meal, but some inevitablycontaminate the oil. Lower levels of glucosinolates in the seed arebelieved to result in lower sulfur content in the oil, and this isbelieved to reduce the objectionable odor characteristics of canola oil(Abraham et al., J. Am. Oil Chem. Soc., 65:392-395, 1988). An analysisof the sulfur content of IMC 01 oil and several generic canola oils hasbeen performed. IMC 01 oil has approximately one-third the sulfurcontent of leading generic canola oils (Table VIII). TABLE VIII SulfurContent of Canola Oils Canola Oils Sulfur Content Acme Brand Canola Oil3.8 ppm Hollywood Brand Canola Oil 3.8 ppm Puritan Brand Canola Oil 3.9ppm IMC 01 Canola Oil 1.3 ppm

[0035] The biochemical, molecular and genetic mechanisms responsible forthe room-odor quality of vegetable oils are not fully understood.Improvements in vegetable oil processing technology, i.e., preferentialremoval of sulfur during processing, less abusive oil extractionprocedures, minimal processing, gentler deodorization, etc., may improvethe overall quality of vegetable oils, including both sensory andfunctional characteristics (Daun et al., J. Am. Oil Chem. Soc.,53:169-171, 1976). IMC 01 will benefit from any such processingimprovements, and will maintain its improved sensory characteristicsover generic canola oil under equivalent processing conditions.

[0036] IMC 01 is true breeding as are its progeny. The traitsresponsible for reduced α-linolenic acid and reduced totalglucosinolates in the seed which yield an oil low in sulfur havingimproved sensory characteristics have a genetic basis. The datapresented herein show that these traits are stable under different fieldconditions. These traits can be removed from the IMC 01 background andare transferred into other backgrounds by traditional crossing andselection techniques.

[0037] Crosses have been made with IMC 01 as one parent to demonstratethat the superior IMC 01 quality/sensory traits are transferred alongwith the superior agronomic traists of another parent such as theCanadian canola line, Westar, into descendents. The parent to which IMC01 is crossed is chosen on the basis of desirable characteristics suchas yield, maturity, disease resistance, and standability. Conventionalbreeding techniques employed in such crossings are well known by thoseskilled in the art. Thus, a method of using the IMC 01 Brassica napus isto cross it with agronomically elite lines to produce plants yieldingseeds having the characteristics listed above.

[0038] The general protocol is:

[0039] a. cross IMC 01 to a selected parent;

[0040] b. produce a “gametic array” using microsphores of the F₁ plantsto produce dihaploid (DH) individuals;

[0041] c. field trial DH₂ individuals for yield and select from IMC 01α-linolenic acid and glucosinolate levels; and

[0042] d. test selected individuals for oil quality using RBD oil.

[0043] Example 3 is a specific example of such work to developdescendents to IMC 01 which retain the desirable quality traits. Thedata of Example 3 show that the quality traits of IMC 01 are heritablein such crosses.

[0044] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that thisExample, while indicating preferred embodiments of the invention, isgiven by way of illustration only. From the above discussion and thisExample, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theinvention to adapt it to various usages and conditions.

EXAMPLE 1

[0045] IMC 01, originally designated DNAP #336, was grown in agreenhouse in Cinnaminson, N.J., over several seasons to select for astable, very low α-linolenic line. Day/night temperatures from Augustthrough December in the greenhouse averaged 80° F./65° F. withfluctuations of ±5° F., 75° F./65° F. from January through April, and85° F./65° F. from March through July. The plants were grown in 1-gallonpots under natural day length, except from October through May when theplants received 14 hours of supplemental lighting. Flowering racemeswere covered with paper bags to prevent cross-pollination, and gentlyshaken to induce seed set. Watering was decreased as pods reachedmaturity.

[0046] For field testing, IMC 01 was planted in multi-location trialsand production plots in Montana, Idaho and Washington. The trials wereplanted in a completely randomized block design with four replications.Each block contained eight plots of 6 meters by 8 rows. IMC 01 was alsoplanted in large acreages (>25 acres) according to standard agronomicprocedures for spring canola production, with a minimum {fraction(1/2)}-mile isolation from other Brassica napus crops. Depending onlocation, the fields were planted in April or May, and harvested inAugust or September. Plantings were made on dryland, following bothfallow or recrop, or under irrigation. Mature pod samples were takenfollowing swathing for chemical analysis.

[0047] For fatty acid analysis, 10-50 seed samples were ground in 15-mLpolypropylene tubes and extracted in 1.2 mL 0.25 N KOH in 1:1ether/methanol. The sample was vortexed for 10 sec and heated for 60 secand in 60° C. water bath. Four mL of saturated NaCl and 2.4 mL ofiso-octane were added, and the mixture was vortexed again. After phaseseparation, 600 μL of the upper organic phase was pipetted intoindividual vials and stored under nitrogen. One μL sample was injectedinto a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 mlength, 0.20 μm df, Bellfonte, Pa.).

[0048] The gas chromatograph was set at 180° C. for 5.5 min, thenprogrammed for a 2° C./min increase to 212° C., and held at thistemperature for 1.5 min. Chromatography settings were: Column headpressure—15 psi, Column flow (He)—0.7 mL/min, Auxiliary and Columnflow—33 mL/min, Hydrogen flow=33 mL/min, Air flow—400 mL/min, Injectortemperature—250° C., Detector temperature—300° C., Split vent—{fraction(1/15)}.

[0049] A standard industry procedure for HPLC analysis of glucosinolateswas used to analyze the glucosinolates composition of the seed (Daun etal., In: Glucosinolate Analysis of Rapeseed (Canola). Method of theCanadian Grain Commission, Grain Research Laboratory, 1981).

[0050] IMC 01 seed was harvested and processed to produce refined,bleached and deodorized (RBD) oil. Some oil was hydrogenated afterrefining, bleaching and deodorization, then redeodorized.

[0051] Before extraction, the seed was tempered to adjust the moisturecontent to 9% and flaked to 0.38 to 0.64 cm in a ribbon blender. Theflakes were cooked in a stack cooker at 82.8° C. for 30 min (8.5%moisture) and pre-pressed with vertical and horizontal bar spacings setto 0.031 cm, vertical shaft speed at 40 rpm and horizontal shaft at 25rpm. The press cake was extracted in a Crown Model 2 extractor at 37.3kg and hexane extracted with a 2:1 solvent to solids ratio.

[0052] The crude oil was alkali refined at 65° C.-70° C. for 30 min with0.2% to 85% phosphoric acid, then mixed with sodium hydroxide toneutralize free fatty acids. Soaps were removed with a water wash (65°C. water, 5 min) and the oils bleached with 0.75% each by weight ofClarion and Acticil bleaching earths for 30 min to remove color bodies.The resulting oil contained no peroxides, 0.08% free fatty acids, andhad a Gardner color of 10-.

[0053] The oil was continuously deodorized at 265° C. at 300 kg/h. Thesteam rate was 1% of feed rate. The deodorized oil was preheated to68-72° C. prior to deaeration. RBD oil was stored in food grade plasticdrums or pails at 4° C. under nitrogen prior to testing.

[0054] For hydrogenation, RBD oil was heated to 350° F. under vacuum ina stainless steel pressure reactor. A 0.5% sulfur-poisoned nickelcatalyst, Englehardt SP-7, was added to the oil at 80.1° C., andhydrogen gas was introduced at 40 psi. Periodic samples were analyzeduntil an oil with a 30.5° C. melting point was achieved. Thehydrogenated oil was redeodorized and stored by the methods describedpreviously.

[0055] The RBD and hydrogenated oil samples were analyzed for room-odorcharacteristics by a trained test panel in comparison with a generic,commercially available RBD canola oil (Proctor & Gamble) and generic,commercially available hydrogenated canola shortening as describedpreviously. The testing protocol used is described in Mounts (J. Am. OilChem. Soc. 56:659-663, 1979) which is hereby incorporated by reference.The testing controlled for temperature of the oil, distance from the oiland room volume, and required that the oil was heated in a separatechamber and pumped into the room containing the trained panelist.

[0056] Specifically, room odor profiles of IMC 01 and a generic canolaoil were obtained as follows:

[0057] A. Room Odor Protocol

[0058] A 150 mL sample of the selected oil was heated to 190° C. for 30min before the start of each panel test. The oil was maintained at thistemperature throughout each session. For each session a fresh oil samplewas used.

[0059] Panelists visited each odor room for approximately 15 sec. A fivemin rest was required between visits. Visitation to each odor room wasrandomized among the panelists.

[0060] The trained panelists judged the room odor for intensity of odor,quality of odor, and odor attributes. The intensity was ranked as: 0-4weak, 5-7 moderate, and 8-10 strong. The quality of odor was judged as:0-1 bad, 2-3 poor, 4-6 fair, 7-8 good, and 9-10 excellent. The odorattributes were ranked as: 0-1 bland, 2-4 weak, 5-7 moderate, and 8-10strong. The flavor attributes were fried, painty, fishy, hydrogenated,burnt, cardboard, metallic, rubbery, waxy, buttery, and nutty.

[0061] B. Generic Oil—IMC 01 Profile Comparison

[0062] A generic, commercially available canola oil (Proctor & Gamble)was used in the IMC room odor tests as the standard or generic canolaoil. In a comparative test, the standard canola oil was significantly(P<0.05) higher in room odor intensity than IMC 01 (Table IX). Thestandard canola oil odor was of “moderate” intensity while the IMC 01was considered “weak”. The overall quality of the IMC 01 room odor wassignificantly (P<0.05) better than the standard canola oil. The standardcanola oil had significantly (P<0.05) higher intensities for fried,painty, and cardboard odors than the IMC 01 oil. TABLE IX Room OdorProfile of Generic (Proctor & Gamble) and IMC 01 Oil Evaluation* IMC 01Generic A. Intensity 3.4^(a) 5.2^(b) B. Quality 5.8^(a) 4.9^(b) C. OdorAttributes Fried 1.9^(a) 2.9^(b) Painty 0.4 1.3 Fishy 0.8^(a) 1.9^(b)Hydrogenated 1.1 0.6 Rancid 0.7 0.9 Burnt 0.8 1.4 Cardboard 0.1^(a)1.5^(b) Metallic 0.5 0.1 Rubbery 0.0 0.0 Waxy 0.6 0.0 Buttery 0.7 0.3

[0063] Pilot plant-processed samples of Example 2 generic canola (lowerucic acid rapeseed) oil and oil from IMC 01 canola with the fatty acidcompositions modified by mutation breeding and/or hydrogenation wereevaluated for frying stability α-linolenic acid contents were 10.1% forgeneric canola oil, 1.7% for canola modified by breeding (IMC 01) and0.8% and 0.7% for IMC 01 oils modified by breeding and hydrogenation.The IMC 01 modified oils had significantly (P<0.05) less room odorintensity that the generic canola oil after initial heating tests at190° C. as judged by a sensory panel under conditions of AOCS Cg 2-83.The generic canola oil had significantly higher intensities for fishy,burnt, rubbery, smoky, and acrid odors than the modified oils. Foamheights of the modified oils were significantly (P<0.05) less than thoseof the generic oil after 20, 30 and 40 hrs of heating and frying at 190°C. The flavor quality of french fried potatoes was significantly(P<0.05) better for all the potatoes fried in modified oils than thosefried in generic canola oil. The potatoes fried in generic canola oilwere described by the sensory panel as fishy. No off-flavors weredetected in potatoes fried in the modified oils.

EXAMPLE 3

[0064] This Example demonstrates that the traits of very low α-linolenicacid and very low glucosinolate content are transferred to IMC 01progeny.

[0065] The pre-production will be crushed and the oil refined forquality.

[0066] Once a canola line has been stabilized, fully conventionalmethods of plant biotechnology, breeding and selection are used tofurther enhance, for example, the agronomic properties of the resultantline in order to improve important factors such as yield; hardiness,etc. Such techniques are also well known and include, e.g., somaclonalvariation, seed mutagenesis, anther and microspore culture, protoplastfusion, etc. See, e.g., Brunklaus-Jung et al., Pl. Breed., 98:9-16,1987; Hoffmann et al., Theor. Appl. Genet, 61, 225-232 (1982), eachherein incorporated by reference).

[0067] A deposit of seed designated IMC 01 has been made in the AmericanType Culture Collection (ATCC) depository (Rockville, Md. 20852) andbears accession number ATCC 40579. The deposit was made on Mar. 2, 1989under conditions complying with the requirements of the Budapest Treaty.

EXAMPLE 4

[0068] IMC 01 was compared to Alto, a “generic” variety of commercialcanola oil.

[0069] Oils were processed using standard commercial refining, bleachingand deodorizing processes. TABLE X Processed Oil Analysis IMC 01 AltoRed color 0.5 0.7 Yellow color 4 5 para-anisidine value 0.97 2.32Peroxide value, meg/g 0.6 0.7 TOTOX value − p-av + 2 (pv) 2.07 3.72Total Polymers, % 0.02 0.01 Total Polar Material, % 0.77 0.36 Free fattyacid, % 0.022 0.013 Fatty acid composition, % C16:0 4.2 4.0 C18:0 2.22.0 C18:l 63.5 62.5 C18:2 22.2 18.3 C18:3 4.9 7.7

[0070] Colors determined by American Oil Chemists' Society (AOCS) methodCc 13b-43, using American Oil Tintometer, Model AF715, The TintometerLTD., Salisbury, England.

[0071] para-Anisidine Value (p-av) determined by AOCS method Cd 18-90,measures secondary oxidation by-producs in oils.

[0072] Peroxide Value determined by AOCS method Cd 8b-90, measures theprimary oxidation product in oils.

[0073] Total polymers determined by AOCS method Cd 22-91, gel-permeationHPLC.

[0074] Total Polar Materials determined by AOCS method Cd 20-91, packedcolumn method adpated to HPLC.

[0075] Free fatty acid determined by AOCS method Ca 5a-40.

[0076] Fatty acid composition determined by AOCS method Ce 1e-91,capillary gas liquid chromatography.

[0077] Oxidative Stability by Oxidative Stability Instrument (OSI)

[0078] Oxidative stability measured by Automatic Oxygen Method (AOM)hours determined by American Oil Chemists Society (AOCS) method Cd12b-92, Fat Stability, Oil Stability Index (OSI), using an OxidativeStability Instrument, manufactured by Omnion/Archer Daniels Midland,Decatur, Ill. IMC 01 Alto AOM hours 26.8 17.5

[0079] Results indicate IMC 01 has greater AOM hours and thereforegreater oxidative stability than Alto.

[0080] Oxidative Stability and Flavor Stability by Accelerated Aging

[0081] The Schaal oven method of accelerated aging is used in the oilindustry to measure the oxidative and flavor stability of oil. TheSchaal oven method involves examining samples of oil at predeterminedintervals held at 60° C. in the dark. One day under Schaal ovenconditions is equivalent to one month storage in the dark at ambienttemperature.

[0082] Oxidative stability is measured by monitoring the increases inperoxides (peroxide value) and secondary oxidative by-products(para-anisidine value) in the oil held at 60° C. for twelve days.

[0083] Flavor stability is determined by a trained sensory panel usingthe same oils tested for oxidative stability.

[0084] Sample Preparation:

[0085] 400 g of oil placed in 500 mL amber glass bottles (80 mm wide,140 mm high, with a 42 mm opening), uncapped, held in 60° C. (range 59to 61° C.) convection oven (Blue M, manufactured by Blue M Electric) for3, 6, 9 and 12 days. One bottle of oil per day per type of oil.

[0086] Samples were frozen immediately after removing from the oven.Peroxide value, para-anisidine value and sensory evaluation were madewithin seven days after samples were frozen.

[0087] Oxidative Stability by Accelerated Aging TABLE XI Changes inPeroxide Value and para-Anisidine Value PV p-AV Days IMC 01 Alto IMC 01Alto 0 .6 0.7 0.97 2.32 3 0.9 5.33 0.99 2.52 6 5.48 11.8 1.36 5.02 910.3 15.7 2.45 6.5 12 14.1 18.8 3.42 7.49

[0088] Flavor Stability by Accelerated Aging:

[0089] Sensory panel trained in evaluation of vegetable oils accordingto protocol of AOCS method Cg 2-83, Flavor Panel Evaluation of VegetableOils. Using AOCS flavor standards panel members were trained to identifythe following off flavors; fishy, green, cardboard, plastic and painty.TABLE XII Overall Acceptance Scores and Total Off-Flavor IntensitiesOverall Acceptance¹ Total Off-Flavors² Day IMC 01 Alto IMC 01 Alto 07.53 6.01 0.59 2.32 3 7.04 3.2 0.87 8.29 6 5.44 3.10 4.17 9.46 9 3.811.78 6.22 10.15 12 4.00 2.00 7.16 12.89

[0090] Correlation between overall acceptance score and totaloff-flavors r²-0.9485 (see FIG. 5).

[0091] Significantly lower overall acceptance scores and lower totaloff-flavor intensities indicates IMC 01 has significantly better flavorstability than Alto.

EXAMPLE 5

[0092] IMC 02 was compared to Stellar, a commercially available lowalpha linolenic variety of canola. TABLE XIII IMC 02 Stellar ProcessedOil Analysis Red color 0.2 0.3 Yellow color 3 3 para-Anisidine value0.48 0.78 Peroxide value, meq/g 0.2 0.6 TOTOX value = p-av + 2(pv) 0.881.98 Total Polymers, % 0.01 0 Total Polar Material, % 0.63 0.42 Freefatty acid, % 0.026 0.014 Fatty acid composition, % C16:0 3.8 4 C18:02.2 2.3 C18:1 66.3 62.8 C18:2 22.3 24.4 C18:3 2.7 3.3 OxidativeStability - pSI AOM Hours AOM hours 31.5 23.1

[0093] Results indicate IMC 02 has greater AOM hours and thereforegreater oxidative stability than Stellar.

[0094] Oxidative Stability by Accelerated Aging TABLE XIV Changes inPeroxide Value and para-Anisidine Value PV p-Av Days IMC 02 Stellar IMC02 Stellar 0 0.2 0.6 0.48 0.78 3 0.7 1.39 0.76 0.84 6 1.5 3.77 0.26 0.999 4.4 7.37 0.82 2.07 12 9.6 13.3 1.18 2.56

[0095] TABLE XV Overall Acceptance Scores and Total Off-FlavorIntensities Overall Acceptance¹ Total Off-Flavors² Day IMC 01 Alto IMC01 Alto 0 8.66 7.75 0.66 0.73 3 6.87 4.18 1.98 7.16 6 7.01 4.57 1.446.07 9 6.10 3.67 2.79 8.41 12 4.16 3.67 5.73 7.83

[0096] Correlation between overall acceptance score and total off-flavorintensities is r²-0.9428 (see FIG. 10).

[0097] Significantly lower overall acceptance scores and loweroff-flavor intensities indicates IMC 02 has significantly better flavorstability than Stellar.

What is claimed is:
 1. A Brassica napus plant comrising seed having atotal glucosinolates content of about 18 μmol/g or less of defatted,air-dried meal; the seed yielding oil having an α-linolenic acid contentof 7% or less relative to total fatty acid content of said seed and asulfur content of less than or equal to 3.0 ppm; and the plant belongingto a line in which these traits have been stabilized for both thegeneration to which the seed belongs and that of its parent generation.2. The seed produced by the plant of claim
 1. 3. The seed produced bythe plant of claim 1 wherein total glucosinate content is about 15μmol/g or less of defatted, air-dried meal.
 4. The oil of the seedproduced by the plant of claim
 1. 5. A Brassica napus plant designatedIMC 01 represented by seed deposited with the ATCC and bearing accessionnumber
 40579. 6. The oil produced from the variety of claim
 5. 7. ABrassica napus seed yielding canola oil having, when hydrogenated,significantly reduced overall room-odor intensity relative to theoverall room-odor intensity of generic canola oil, a significantdifference in overall room odor-intensity indicated by a difference ofgreater than 1.0 obtained in a standardized sensory evaluation.
 8. ABrassica napus comprising oil, which when non-hydrogenated, issignificantly reduced in fishy odor intensity relative to the fishy odorintensity of generic canola oil, a significant difference in fishy odorintensity indicated by a difference of greater than 1.0 obtained instandardized sensory evaluation.
 9. A Brassica napus plant wherein atleast one parent was the variety of claims 1 or
 5. 10. The progeny ofthe plant of claims 1, 5 or
 9. 11. A plant produced from the crossing ofIMC 01 with an agronomically elite variety of Brassica napus, the plantyielding a seed having a total glucosinolates content of about 18 μmol/gor less of defatted, air-dried meal, said seed yielding extractable oilhaving (1) an α-linolenic acid content of about 7% or less relative tototal fatty acid content of said seed, and (2) a sulfur content of lessthan or equal to 3.0 ppm.
 12. The plant of claim 11, wherein theagronomically elite parent is the Canadian canola line, Westar.
 13. Aprocess for producing a canola of enhanced commercial utilitycomprising: (a) crossing the Brassica napus IMC 01 with an agronomicallyelite variety; (b) selecting the off-spring of step (a) which yield aseed having a total glucosinolates content of about 18 μmol/g or less ofdefatted, air-dried meal, said seed yielding extractable oil having (1)an α-linolenic acid content of about 7% or less relative to total fattyacid content of said seed, and (2) a sulfur content of less than orequal to 3.0 ppm.
 14. The oil extracted from the seed produced by theprocess of claim
 13. 15. A method of using the Brassica napus IMC 01comprising: (a) crossing IMC 01 with an agronomically elite variety; (b)selecting the off-spring of step (a) which yield a seed having a totalglucosinolates content of about 18 μmol/g or less of defatted, air-driedmeal, said seed yielding extractable oil having (1) an α-linolenic acidcontent of about 7% or less relative to total fatty acid content of saidseed, and (2) a sulfur content of less than or equal to 3.0 ppm; (c)producing sufficient progeny of the seed selected in step (b) to extractoil.
 16. The Brassica napus designated HW 3.001, a progeny line of thecross of IMC 01 with Westar.
 17. An improved vegetable oil extractedfrom Brassica napus seeds, said seeds having: (1) an oil which exhibitsfollowing crushing and extraction (a) an α-linolenic acid content of 7%or less relative to total fatty acid content of said seed; (b) a sulfurcontent of less than or equal to 3.0 ppm; and (2) a total glucosinolatescontent of about 18 μmol/g or less of defatted, air-dried meal.
 18. Theoil produced from the progeny of claim 1, 5 or 9, as described in claim10, wherein the stability of such oil measured in AOM hours is fromabout 25.0 to about 35.0.
 19. The oil as described by claim 18 whereinthe stability in AOM hours is from 26.8 to 31.5.